The active sites of a number of metalloenzymes contain a coupled binuclear copper unit which exhibits nearly constant chemical and spectroscopic properties and interacts with oxygen as part of its biological function. This copper site is found in metalloproteins which perform vital roles in copper and iron metabolism, oxygen activation, and electron transfer. In different proteins, this site cooperatively binds oxygen (hemocyanin (Hc)), hydroxylates monophenols (tyrosinase) and in conjunction with other copper centers, couples four 1-electron oxidations of substrate to the 4-electron reduction of dioxygen to water (laccase, ceruloplasmin and ascorbic acid oxidase). The objective of this proposed research is to study in detail the chemistry and spectroscopy of this binuclear site to understand its unique spectral features and thus its electronic and geometric structure and the correlation of these features with protein function. We have generated a series of derivatives which allow the active site to be systematically varied and studied through a variety of appropriate spectroscopic methods. These studies have led to the development of a model of the oxy Hc active site. Our studies have shown that the binuclear copper site in tyrosinase is extremely similar to that in Hc. However, in tyrosinase, substrate analogues also bind with high affinity to the copper site producing unusual copper spectral features. Analysis of these features has thus led to significant insight into the mechanism of this enzyme on a molecular level. Studies on the binuclear copper site in laccase (called Type 3) have demonstrated that there are important differences relative to that in Hc and tyrosinase. In particular, exogenous ligands cannot bridge the two coppers at the Type 3 site, but instead bridge to an additional copper (Type 2) center, thus indicating the presence of a trinuclear copper cluster in laccase. We propose to investigate this trinuclear copper site, its binding of exogenous ligands, its reaction with oxygen and its interaction with the Type 1 copper, and to determine the correlations between these copper centers in laccase and those in the more complicated milticopper oxidases. Further, since we now have a reasonable understanding of the interaction of O2 with Hc and tyrosinase, we propose to proceed with experiments directed to 1)- understanding the nature of the peroxide copper bond and its activation toward oxygenation and reduction. 2)- probing exogenous and endogenous ligand interactions with the deoxy site in complement to recent crystallographic results on this form, and 3)- using a "spectral probe" derivative of the Hc biopolymer, which we have prepared to study the effects of cooperative interactions at an active site level.